Reagentless, reusable, ultrasensitive electrochemical molecular beacon aptasensor
Reagentless, reusable, ultrasensitive electrochemical molecular beacon aptasensor
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DOI：
10.1021/ja053121d
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发表时间：
2006-01-11
影响因子：
15
通讯作者：
O'Sullivan, CK
中科院分区：
文献类型：
--
作者：
Radi, AE;Sánchez, JLA;O'Sullivan, CK
合成了具有氧化还原活性Fc片段和适体链末端巯基的凝血酶结合适体的双功能衍生物。二茂铁标记的适体硫醇通过S-Au键在多晶金电极表面自组装，表面被2-巯基乙醇阻断，形成混合单层。利用荧光分子信标，确定了反离子对四联体形成的影响。采用循环伏安法（CV）、差分脉冲伏安法（DPV）和电化学阻抗谱（EIS）对配体修饰电极进行了电化学表征。在pH 8.0的10 mM N-（2-羟乙基）哌嗪-N-2-乙磺酸（HEPES）缓冲液中，固定在电极表面的二茂铁基部分发生一步氧化还原反应，显示出伏安信号。2-巯基乙醇阻断后，DPV电流信号明显增加，有效去除非特异性吸收的适配体，而不是结合到电极表面，或者由于适配体-凝血酶亲和相互作用的形成。阻抗测量结果与差分脉冲伏安法（DPV）一致，显示法拉第电阻在相同的顺序上降低。凝血酶结合的“信号开启”可归因于探针修饰膜上的构象从随机线圈状结构改变为四重结构。改性电极的DPV在5.0 ~ 35.0 nM范围内对Fc氧化信号呈线性响应，线性相关r = 0.9988，检出限为0.5 nM。通过在1.0 M HCl中展开适体，分子信标适体传感器可以完全再生，并且在随后的凝血酶结合后可以再生25次而没有电化学信号损失。
A bifunctional derivative of the thrombin-binding aptamer with a redox-active Fc moiety and a thiol group at-the termini of the aptamer strand was synthesized. The ferrocene-labeled aptamer thiol was self-assembled through S-Au bonding on a polycrystalline gold electrode surface and the surface was blocked with 2-mercaptoethanol to form a mixed monolayer. By use of a fluorescent molecular beacon, the effect of counterions on quadruplex formation was established. The aptamer-modified electrode was characterized electrochemically by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The modified electrode showed a voltammetric signal due to a one-step redox reaction of the surface-confined ferrocenyl moiety of the aptamer immobilized on the electrode surface in 10 mM N-(2-hydroxyethyl) piperazine-N-2-ethanesulfonic acid (HEPES) buffer of pH 8.0. An increase in the DPV current signal was evident after blocking with 2-mercaptoethanol, effectively removing aptamer nonspecifically absorbed rather than bound to electrode surface or due to the formation of the aptamer-thrombin affinity interaction. The impedance measurement, in agreement with the differential pulse voltarnmetry (DPV), showed decreased Faradaic resistances in the same sequence. The "signal-on" upon thrombin association could be attributed to a change in conformation from random coil-like configuration on the probe-modified film to the quadruplex structure. The DPV of the modified electrode showed a linear response of the Fc oxidation signal to the increase in the thrombin concentration in the range between 5.0 and 35.0 nM with a linear correlation of r = 0.9988 and a detection limit of 0.5 nM. The molecular beacon aptasensor was amenable to full regeneration by simply unfolding the aptamer in 1.0 M HCl, and could be regenerated 25 times with no loss in electrochemical signal upon subsequent thrombin binding.